Biologically active materials

ABSTRACT

A polymer-drug conjugate, in which the polymer is the polysaccharide dextrin, linked directly or indirectly to the drug, is effective to deliver the drug to a target site and is biodegradable. The conjugate may be prepared by succinoylating dextrin followed by reaction with the drug or a derivative thereof.

This appln is a 371 of PCT/GB98/01567 filed Jun. 12, 1998, which claimsbenefit of Prov. No. 60/054,277 filed Jul. 30, 1997.

FIELD OF THE INVENTION

This invention relates to biologically active materials, and, inparticular, to materials which comprise a polymer linked to abiologically active agent. The invention is concerned with materialsknown as polymer-drug conjugates which typically contain a therapeuticagent, in particular, an anti-cancer drug, linked to a polymer backbone.The linkage between the polymer and the drug is by covalent bonding.

BACKGROUND OF THE INVENTION

In designing a polymer-drug conjugate, the aim is to deliver a drugeffectively to a therapeutic site such as a tumour. It is known, forinstance, that polymer-drugs given intravenously can accumulateselectively in solid tumour tissue by the EPR effect.

The most commonly used anticancer agents are low molecular weightcompounds which readily gain access to cells by rapid passage across thecell membrane. After intravenous (IV) administration, a large percentageof the injected dose leaves the circulation within a few minutes,resulting in a ubiquitous body distribution of drug and little selectiveconcentration in tumour tissue. By creating a macromolecularpolymer-anticancer drug conjugate, there is provided an opportunity toimprove tumour specific targeting, to minimise drug entry into sites oftoxicity, to control precisely the rate of drug liberation at the targetsite (giving opportunities for long-term controlled release) and deliverthe active principal intracellularly, providing a means to overcomep-glycoprotein related multidrug resistance.

Numerous polymers have been proposed for synthesis of polymer-drugconjugates including polyaminoacids, polysaccharides such as dextran,and synthetic polymers such as N-(2-hydroxypropyl)methacrylamide (HPMA)copolymer. However, these polymers have limitations. For example, adextran-doxorubicin conjugate has been tested clinically and been foundto be much more toxic than the parent drug and the HPMA copolymers whichhave been clinically tested have the disadvantage of beingnon-biodegradable in the main chain.

STATEMENTS OF INVENTION

The present invention provides a polymer-drug conjugate in which thepolymer is the polysaccharide dextrin. The polymer-drug conjugate may beone in which the polymer is linked directly to the drug or one in whichthe polymer is linked indirectly to the drug, for instance, by means ofa “biodegradable spacer” to which both the drug and the polymer arelinked. The dextrin is preferably a non-cyclic dextrin.

It has been found that dextrin is not only a suitable material forforming a biocompatible polymer-drug conjugate capable of delivering adrug to a target site and of releasing the drug at such a site but isalso biodegradable in a manner such that it may be used at a molecularweight which is suitable for the particular drug and its applicationwithout any upper limit imposed by the need to ensure excretion of thepolymer.

The term “dextrin” means a glucose polymer which is produced by thehydrolysis of starch and which consists of glucose units linked togetherby means mainly of alpha-1,4 linkages. Typically dextrins are producedby the hydrolysis of starch obtained from various natural products suchas wheat, rice, maize and tapioca. In addition to alpha-1,4 linkages,there may be a proportion of alpha-1,6 linkages in a particular dextrin,the amount depending on the starch starting material. Since the rate ofbiodegradability of alpha-1,6 linkages is typically less than that foralpha-1,4 linkages, for many applications it is preferred that thepercentage of alpha-1,6 linkages is less than 10% and more preferablyless than 5%.

Any dextrin is a mixture of polyglucose molecules of different chainlengths. As a result, no single number can adequately characterise themolecular weight of such a polymer. Accordingly various averages areused, the most common being the weight average molecular weight (Mw) andthe number average molecular weight (Mn). Mw is particularly sensitiveto changes in the high molecular weight content of a polymer whilst Mnis largely influenced by changes in the low molecular weight of thepolymer.

It is preferred that the Mw of the dextrin is in the range from 1,000 to200,000, more preferably from 2,000 to 55,000.

The term ‘degree of polymerisation’ (DP) can also be used in connectionwith polymer mixtures. For a single polymer molecule, DP means thenumber of polymer units. For a mixture of molecules of different DP's,weight average DP and number average DP correspond to Mw and Mn. Inaddition DP can also be used to characterise a polymer by referring tothe polymer mixture having a certain percentage of polymers of DPgreater than a particular number or less than a particular number.

It is preferred that, in the dextrin-drug conjugate of the presentinvention, the dextrin contains more than 15% of polymers of DP greaterthan 12 and, more preferably, more than 50% of polymers of DP greaterthan 12.

The drug loading on the polymer is preferably from 0.5 to 99.5 mole %.

A targeting group may be attached either directly or indirectly to thepolymer of the conjugate. It is preferred that the ratio of drug totargeting group is from 1:99 to 99:1.

Preferably the dextrin used in a dextrin-drug conjugate of the presentinvention is water soluble or at least forms a suspension in water.

The dextrin of a dextrin-drug conjugate of the invention may be in theform of either unsubstituted dextrin (as obtained by the hydrolysis ofthe starch) or may be substituted by one or more different groups. Thesubstituents may be negatively charged groups, for instance, sulphategroups, neutral groups or positively charged groups, for instance,quaternary ammonium groups. In the case where the subsistent group issulphate, it is preferred that the sulphated polysaccharide contains atleast one sulphate group per saccharide (glucose) unit. A particulardextrin sulphate is dextrin-2-sulphate.

Examples of drugs which may form suitable conjugates with dextrin are:-alkylating agents such as cyclophosphamide, melphalan and carmusline;antimetabolites such as methotrexate, 5-fluorouracil, cytarabine andmercaptopurine; natural products such as anthracyclines (egdaunorubicin, doxorubicin and epirubicin), vinca alkaloids (egvinblastine and vincristine) as well as dactinomycin, mitomycin C,taxol, L-asparaginase and G-CSF; and platinum analogues such ascisplatin and carboplatin.

The present invention also provides a pharmaceutical compositioncomprising a dextrin-drug conjugate and a pharmaceutically acceptableexcipient or diluent therefor.

In addition, the present invention provides the use of a polymer-drugconjugate of the invention in the treatment of a medical condition inconnection with which the drug is effective, Furthermore, the inventionprovides the use of a polymer-drug conjugate of the invention in themanufacture of a medicament for use in the treatment of a medicalcondition in connection with which the drug is effective.

The present invention also provides a method of treating an animalsubject, including a human being, including treating the animal subjectwith a pharmaceutically effective dose of a dextrin-drug conjugate. Theconjugate may be administered by any appropriate method, for instance,intravenously, intraperitoneally, orally, parentally or by topicalapplication.

Various methods have been proposed for the preparation of polymer-drugconjugates. Within the scope of the present invention is a methodcomprising succinoylating dextrin and reacting the succinoylated dextrinwith a drug or reactive derivative thereof.

Preferably, the dextrin is dissolved in anhydrous dimethyl formamide andis contacted with dimethyl amino pyridine and succinic anhydride. Theresultant mixture is then purged with an inert gas and chemical reactionis allowed to take place over a prolonged period, preferably at least 12hours.

The resultant succinoylated dextrin is reacted with a drug or drugderivative eg. doxorubicin hydrochloride to form the polymer-drugconjugate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical illustration of the effect of dextrins on redblood cell lysis.

FIG. 2 is a graphical illustration of the effect of compounds MLD 15/73,MLD 5/29 and DXO 40G against Poly L lysine and Dextran.

FIGS. 3A and 3B illustrate the synthetic route for the preparation ofdextrin-doxorubicin.

FIG. 4 is a graphical illustration of the cytotoxicity of doxorubicinand a dextrin-dox conjugate against L1210 cells (72 h).

FIG. 5 is a graphical illustration of tumour levels of doxorubicin afteradministration of dextrin-doxorubicin and free doxorubicin.

FIG. 6 illustrates an electrophoresis curve for the crude and purifiedradiolabelled dextrin-tyrosinamide.

FIGS. 7A, 7B and 7C illustrate body distribution of 125I-labelleddextrin (MW 51000) at 5 minutes, 30 minutes and 1 hour respectively.

FIG. 8 illustrates the elution of radioactivity from a PD10 Column(Sephadex G-25).

FIG. 9 illustrates the stability of dextrins of different molecularweight in Saline Phosphate Buffer at pH 7.4 without enzymes.

FIG. 10 illustrates the stability of dextrins of different molecularweight in Salt Citrate Buffer at pH 5.5 without enzymes.

FIG. 11 illustrates DX0 40G degradation and low Mw saccharides releaseprofiles during incubation in rat plasma at 37 C.

FIG. 12 illustrates MLD 15/73 degradation and low Mw saccharides releaseprofiles in rat plasma at 37 C.

FIG. 13 illustrates degradation of dextrin MD 15/73.

EXAMPLES OF THE INVENTION

The present invention will now be further described with reference tothe following examples. In these examples three dextrin materials areused, their molecular weight characteristics being as follows (Table 1):

TABLE 1 Examples confirming the Biocompatibility of Dextrins Code Mw MnMw/Mn  MLD5/29  5K  6K 2.74 DX04/0G 15K  6K 2.74 MLD15/73 51K 28K 1.84

Example 1 Effect of Dextrins on the Stability of Rat Red Blood CellsIncubated in Vitro.

Method

Dextrins of different molecular weight were incubated with isolated ratred blood cells (RBC) and haemoglobin release detectedspectrophotometrically. Various dilutions of the test polymers(dextrins) and of the reference polymer polyethyleneimine were made upin PBS and mixed with an equal volume of fresh 2% w/v suspension of ratRBCs. This preparation was centrifuged at 1500×g for 10 min at roomtemperature. Then, 100 μl of the supernatant was removed and the levelsof haemoglobin quantitated spectrophotometrically at 5.50 nm. Theresults illustrated in FIG. 1 were expressed as % lysis relative to thatof a Triton X-100 control (Sigma) which gave 100% RBC lysis.

Results

None of the dextrin polymers induced red cell lysis. Polyethyleneimineand the detergent Triton X-100 (control 100% lysis) both causedhaemoglobin release.

Example 2 Cytotoxicity of Unmodified Dextrins Against L1210 Cells.

Method

L1210 cells were seeded at a density of 1×10⁴ cells per well into 96well ‘v’ bottomed microtiter plates (Costar) in RPMI 1640 tissue culturemedia (Gibeo) supplemented with 10% Foetal Calf Serum (FCS) (Gibeo). Allincubations were carried out at 37° C. in an atmosphere of 5% CO₂.

The cells were then left for 24 h. Prior to being incubated with thepolymers, the cells were centrifuged at 1000×g for 10 minutes at roomtemperature. This served to pellet the cells and allow the removal ofthe media. The polymers were then dissolved in RPMI 1640 containing 10%FCS and filter sterilised through 0.2μ filters (Aerodisk). Followingthis, the pellet was resuspended in media containing the appropriateconcentration of the dextrins of different molecular weight. Positiveand negative reference controls were incorporated, dextran Mw 72 000(Sigma) as a negative control and poly(L-Lysine) Mw 56 500 (Sigma) as apositive control. These polymers were added to cells at the sameconcentrations as the test polymers. The tetrazolium dye MTT was used toassess cell viability. The cells were then incubated for 67 h prior tothe addition of the MTT. After a further incubation period of 5 h, thecells in suspension were subjected to centrifugation, the media removedand 100 μl of optical grade DMSO (Sigma) added. The plates were read at550 mn using a Titerteck plate reader. The results are illustrated inFIG. 2. The % viabilities are optical densities (OD's) expressed as a %of the OD seen in cell cultures containing no polymer.

Results

None of the dextrins was cytotoxic. The toxic effect of the referencecontrol poly-L-Lysine can be seen.

Examples Relating to Dextrin Modification for Coupling of Pendent Groups

To bind pendent drugs or side chains to dextrins there are a number ofpossible activation methods that may be used. These include:

a) Periodate Oxidation

This is a ring opening method resulting in polyaldehyde derivatives of apolysaccharide (Bruneel and Schacht 1992). Unfortunately this reactionis prone to a number of side reactions that makes the production ofsingle, pure, easily characterised compound quite difficult. Inparticular there is evidence for the formation of intra-residualhemiacetal formation. However, when formed aldehyde groups representsites where amines can be attached to the polymer in the form of Schiffbase structures or alkyl amines.

b) The 4-nitrophenyl Chloroformate Method

This is easily carried out at 0° C. and there is much informationavailable on the analysis of the various groups that do, or do not,react. Clean up/purification procedures would appear to bestraightforward and percentage reactivity of active groups in the rangeof interest would appear to be controllable to an effective extent.There is some concern, however, over the formation of undesirable fivemembered cyclic carbonates (Bruneel and Schacht 1992).

c) Succinoylation

Activation by succinoylations is also an easily performed operation. Theconditions employed are however somewhat acerbic by comparison with thechloroformate method. The initial reaction requires refluxing at 40° C.for 24 hr in dry DMSO, but results in the formation of carbonatefunctionalities. This reaction also includes an extra step on the way tothe production of the amine derivative by virtue of going through anintermediate step involving N,N′-carbonyldiimidazol. Here too,purification would appear to be relatively uncomplicated.

d) Cyanogen Bromide Activation

Activation by cyanogen bromide is easily carried out in aqueousconditions at room temperatures. (Immobilised Affinity LigandTechniques, Herman GT, Mallia AK and Smith PK. Pub: Academic Press, NewYork, 1992). Excess reagent must however be neutralised and eliminatedas it may liberate hydrogen cyanide in an acidic environment. Inaddition to this side reactions may occur resulting in several reactiveintermediates. It does however have advantages in that a primary aminecan be introduced directly on the reactive intermediate. Finally thereis evidence for the linkage being unstable resulting in slow release ofthe pendant group which may or may not be advantageous or desirable.

Examples Modification of Dextrins by Succinoylation and Reaction withCompounds Containing Amino Groups ( in relation to (c) above).

All three dextrin samples were modified by means of succinoylation.Initially, attempts to modify the samples to the extent of 50, 20, 10,5, 2.5 and 1 mol% were carried out. The sample with the highestmolecular weight was taken forward for further reaction withtyrosinamide and doxorubicin.

Example 3.1 Synthesis of Succinoylated Dextrins

In triplicate batches of dextrin (1 g, MD 15/73, 1.94×10⁻⁵ mol) wasdissolved in anhydrous dimethyl formamide (DMF, 10 m). Dimethyl aminopyridine (DMAP, 28.3 mg) was then added followed by succinic anhydride(62 mg, 6.2×10⁻¹ mol, 10 mol %). The mixture was then purged withnitrogen, sealed and left to stir for 24 h at temperatures of 20, 30, 40and 50° C. Following this the reaction mixture was poured onto rapidlystirring diethyl ether (100 ml) and magnetically stirred overnight. Theether was then removed by filtration under vacuum and the resultingsolid dissolved into a minimum of distilled water poured into a dialysismembrane (Visking tubing, MW cut-off 12000-1400) and purified bydialysis against 2×2L of distilled water. The resulting solution wasfreeze dried and recovery calculated. Degree of succinoylation was thedetermined by titration against a relevant standard of NaOH. Afterapplication of the students T-Test there was no difference in the degreeof succinoylation for any reaction condition. Results are shown in Table2.

TABLE 2 Results of the effect of temperature on the succinoylation ofdextrin Mol % % Recovery Succinoylation Temperature (g) (± SD) (± SD)  20 * ND ND 30 53.3  (6.7) 6.64 (0.74) 40 73.4 (10.7) 5.49 (0.32) 50 42.6(12.3) 2.26 (0.12) * Did not dissolve

As an alternative procedure the product may be purified by gelfiltration, ultrafiltration or centrifugation against a pore controlledmembrane.

The same reaction conditions (adjusting the ratios of the components orsolvents) were used to achieve degrees of succinoylation of the dextrinsamples of different molecular weights (MW 4950, 15000 and 51,100). SeeTable 1.

Example 3.2 Conjugation of Doxorubicin to Succinoylated Dextrins

Succinoylated dextrin (51 mg, 1×10⁻⁶ mol, MD 15/73, 5.3 mol %succinoylation) was dissolved in 2 ml of distilled water, to this wasadded EDC (3.51 mg 1.8×10⁻⁵ mol). This was allowed to react at roomtemperature while gently stirring for 30 min. After this timeDoxorubicin hydrochloride (Pharmacia) was added (9.67 ml of a 1 mg ml⁻¹solution. 1.67×10⁻⁵ mol). The reaction vessel was wrapped in foil andallowed to stir overnight at room temperature. The product was isolatedby solvent extraction with chloroform and passage over a LH 20 superdexcolumn (Pharmacia) followed by freeze drying to constant weight. Finallyfree and conjugated Doxorubicin was measured by extractive HPLC. Resultsof various conjugations are summarised in Table 3.

TABLE 3 Incorporation of Doxorubicin on various Dex-Dox ConjugatesSuccinoylation Coupling Percent W/W Mol % Conditions Time incorporation0.5  EDC/Water Overnight  0.127 6.53 EDC/Water Overnight 4.2 6.53CDI/DMF Overnight 3.9 5.3  CDI/DMF 4 hours 2.1 14.9 EDC/water/Sulpho-NHS 4 hours  6.39 14.9  EDC/WATER 4 hours  2.82

Alternatively Doxorubicin may be conjugated using, Sulpho-NHS and EDC inaqueous conditions to improve coupling yields or with other peptitdecoupling agents and solvent of choice.

The synthetic route for the preparation of dextrin-doxorubicin isillustrated in FIGS. 3a and 3 b.

Example 3.3 Conjugation of Tyrosinamide to Succinoylated Dextrin

Succinoylated dextrin (57.7 mg, MD 15/73, 1 mol%) was dissolved in 3 mlof DMF, to this was added CDI (10.4 mg, 6.4×10⁻⁵ mol) dissolved in DMF(1 ml). This was allowed to react at 25° C. while stirring for 1 h.After this time tyrosinamide (97 mg, 8.96×10⁻⁴ mol was dissolved in DMF(1 ml) and added to the reaction. This was allowed to react stirring atroom temperature at 25° C. for 48 h. The product was isolated by removalof the DMF by rotary evaporation. To the product was added water (3 ml)followed by freeze drying until constant weight was maintained. Theproduct was purified by dialysis against distilled water (24 h).

Example 3.4 Conjugation of Biotin Hydrozide to Succinoylated Dextrin

Succinoylated dextrin (57.7 mg, MD 15/73, 1 mol %) was dissolved in 3 mlof DMF, to this was added CDI (10.4 mg, 6.4×10⁻⁵ mol) dissolved in DMF(1 ml). This was allowed to react at 25° C. while stirring for 1 hour.After this time biotin hydrozide (97 mg, 8.96×10⁻¹ mol was dissolved inDMF (1 ml) and added to the reaction. This was allowed to react stirringat room temperature at 25° C. for 48 hour. The product was isolated byremoval of the DMF using by rotary evaporation. To the product was addedwater (3 ml) followed by freeze drying until constant weight wasmaintained. The product was purified by dialysis against distilled water(24 h).

Example 4 Evaluation of the Cytotoxicity of Doxorubicin andDextrin-doxorubicin Conjugates

Method

L1210 cells were seeded at a density of 1×10⁴ cells per well into 96well ‘v’ bottomed microtiter plates (Costar) in RPMI 1640 tissue culturemedia (Gibeo) supplemented with 10% Foetal Calf Scrum (FCS) (Gibeo). Allincubations were carried out at 37° C. in an atmosphere of 5% CO₂.

The cells were then left for 24 h. Prior to being incubated with thepolymers the cells were centrifuged at 1000×g for 10 minutes at roomtemperature. This served to pellet the cells and allow the removal ofthe media.

Doxorubicin and dextrin doxorubicin were then dissolved in RPMI 1640containing 10% FCS and filter sterilised through 0.2μ filters(Aerodisk). Following this, the pellet was resuspended in mediacontaining the appropriate concentration of drug or drug conjugate.

The tetrazolium dye MTT was used to assess cell viability. The cellswere incubated for 67 h prior to the addition of the MTT. After afurther incubation period of 5 h, the cells in suspension were subjectedto centrifugation, the media removed and 100 μl of optical grade DMSO(Sigma) was added. The plates were read at 550 mn using a Titerteckplate reader. Results (OD) were expressed as a % of the OD seen in cellcultures containing no polymer. They are illustrated in FIG. 4.

Results

It can be seen that both doxorubicin and the dextrin doxorubicinconjugate (prepared in example 3.2) were toxic to L1210 cells in vitro.This confirms that active doxorubicin can be released from the conjugateover the 72 h incubation period. It is not surprising that the conjugateis less active than the parent drug in vitro. This is a well knownphenomenon attributed to the very slow capture of a polymeric drug bycells by the mechanism of endocytosis. Free doxorubicin penetrates thecell immediately by passing across the cell membrane.

Example 5 Experiments to Demonstrate the Targeting onDextrin-doxorubicin to sc. B16F10 (mouse melanoma)

Method

C57 black mice were injected subcutaneously (sc) with 10⁵ B16F10melanoma cells in normal saline (100 μl). The mice were checked dailyfor the appearance of tumours and when palpable (10-13 days) the micewere then injected iv with dextrin-doxorubicin (6.0 wt % dox) at a doseof 5 mg/kg dox-equivalent. The mice were culled after 1 min, 1 and 24 hand the tumours were removed, weighed and homogenised in doubledistilled water (2 ml). The total doxorubicin content was measured byHPLC after acid hydrolysis and extraction into 4:1 chloroform:isopropylalcohol. Doxorubicin content was then expressed as μg dox/per g oftumour.

Results

After administration of dextrin-doxorubicin no signs of toxicity wereobserved. It can be seen that administration of drug as a dextrinconjugate resulted in significantly higher tumour levels than seenfollowing administration of free drug (FIG. 5).

Example 6 Radiolabelling of Dextrin-tyrosinamide

Method

To allow monitoring of body distribution and rate of degradation thehighest molecular weight dextrin-TyrNH2 was labelled using theChloramine T method.

Dextrin-tyrosinamide (5 mg) was dissolved in phosphate buffer (0.5 ml.0.05 M. pH7.0. Na[¹²⁵I]iodide (5 μl, 5 mCi) was added to the reactionvessel and was left stirring for 2 min. Chloramine T (75 μl, 2 mg/ml)was added and left stirring for 15 min. Sodium metabisulphate (500 μl, 2mg/ml) and a crystal of potassium iodide was added to the reactionvessel and left for 2 min.

Specific activity and purity was determined by paper electrophoresis.Whatman chromatography paper (5×30 cm strips) were divided into 5 mmstrips by pencil (40 strips). Marking the fifth strip as the point ofapplication for the sample. The paper was soaked in barbitone buffer(Sigma B6632) and then blotted dry. Into a paper electrophoresis tank(Shandon) was placed the same buffer and the paper was placed on thesupporting bars. Na[¹²⁵I]iodide (4 μl) was placed on the applicationpoint nearest the anode as a reference and the crude and pureradiolabelled polymer (4 μl) was placed on the application point nearestthe anode as a reference and the crude and pure radiolabelled polymer (4μl) was loaded onto separate strips. The tank was connected to a powersupply and the samples run for 30 min at 400V (constant voltage). Thechromatography papers were removed and each 5 mm strip was cut, andplaced into counting tubes with 1 ml of water in each and the presenceof radioactivity was measured using a gamma counter (Packard). Theresults were plotted as counts per minute against distance migrated.Results are illustrated in FIG. 6.

Results

The dextrin product was radiolabelled and had a specific activity ofapproximately 20 μCi/mg.

Example 7 Body Distribution of ¹²⁵I-Labelled Dextrin

Adult male Wistar rats were anaesthetised with a mixture of oxygen andisoflurothane and radiolabelled dextrin-tyrosinamide (100 μl, specificactivity 11.8 μCi/mg) were injected iv, ip or subcutaneous). The ratswere culled after the desired time using carbon dioxide. The rat wasweighed and all major organs (lungs, heart, liver, kidneys, spleen,thyroid, urine, bladder or blood) were removed. After ip administrationa peritoneal wash was also taken. The organs were homogenised in waterand samples (1 ml in triplicates) were counted in the gamma counter. Theresults expressed as % dose injected for each organ are illustrated inFIGS. 7a, 7 b and 7 c.

Samples of the urine were subjected to gel permeation chromatography(GPC) using a PD10 column to evaluate the degradation of the polymer inthe body. Results are shown in FIG. 8.

Results

After 1 h most of the radioactivity recovered following both ip and ivinjection was detected in the urine and faeces. GPC analysis of urinesamples indicated liberation of low molecular weight degradationproducts.

Example 8 Evaluation of the Rates of Hydrolytic and EnzymaticDegradation of Dextrin Samples of Different Molecular Weight

Experiments were carried out to determine the stability of the threedextrin samples in physiological buffers (pH 7.4 and 5.5) and also inthe presence of enzymes (plasma and lysosomal enzymes (tritosomes).

The molecular weight of the samples and changes with time were monitoredby gel permeation chromatography. Two types of experiment wereundertaken.

Example 8.1 Degradation of Native and Modified Non-radiolabelledDextrins by Enzymes followed by HPLC/GPC with RI Detection

Method

GPC size exclusion was carried out using a TSK G5000 PW and G2000 PWcolumns in series (7.5×300 mm). The sample injection loop was 20 μl, themobile phases used were saline phosphate buffer (PBS) at pH 7.4 andlater high salt (NaCl 0.25M) citrate buffer at pH 5.5 made with doubledistilled water in both cases. The eluant flow rate in all cases was 1ml min¹.

Column calibration was carried out with pullulan and glucose molecularweight standards. The method of detection was by means of differentialrefractometry using a Knauer refractive index detector.

Samples (at concentration of approximately 5 mg ml⁻¹) were incubated inthe appropriate buffer (with or without enzymes) for several days at 37°C. At various time periods a small sample was taken and subjected toGPC.

Results

In the absence of enzyme, the dextrin samples at 37° C. did not show anyevidence of hydrolysis. FIGS. 9 and 10 illustrate the stability ofdextrin of different molecular weights in buffers without enzymes. Whenplasma enzymes were added samples degraded rapidly over the first hourand more slowly thereafter. Addition of lysosomal enzymes did not causedextrin degradation. FIGS. 11 and 12 illustrate for samples DX0409 andMLD15/73 respectively, the dextrin degradation and low molecular weightsaccharides release during incubation in rat plasma at 37° C.

Example 8.2 Degradation of ¹²⁵I-labelled Dextrin Measured by PD10 GPC

Method

The radiolabelled dextrin sample prepared for the body distributionstudies was also incubated with plasma and lysosomal enzymes and samplessubjected to PD10 Sephadex chromatography to evaluate the rate ofdegradation. Fractions eluting from the column were assayed forradioactivity and the results expressed as the percentage decrease inpeak one (the polymer peak). The results for dextrin sample MD15/73 areillustrated in FIG. 13.

Results

Although the polymer sample degraded rapidly in the presence of plasmaenzymes the product appeared stable in the presence of lysosomalenzymes.

What is claimed is:
 1. A polymer-drug conjugate in which the polymer isthe polysaccharide dextrin which is covalently linked either directly orindirectly to the drug and the drug is doxorubicin.
 2. A polymer-drugconjugate in which the polymer is the polysaccharide dextrin which iscovalently linked either directly or indirectly to the drug and thedextrin is a non-cyclic dextrin.
 3. The polymer-drug conjugate accordingto claim 1 or 2 wherein a percentage of alpha-1,6 linkages in thedextrin is less than 10%.
 4. The polymer-drug conjugate according toclaim 3 wherein the percentage of alpha-1,6 linkages in the dextrin isless than 5%.
 5. The polymer-drug conjugate according to claim 1 or 2wherein a weight average molecular weight of the dextrin is in the rangefrom 1,000 to 200,000.
 6. The polymer-drug conjugate according to claim5 wherein the weight average molecular weight of the dextrin is in therange from 2,000 to 55,000.
 7. The polymer-drug conjugate according toclaim 1 or 2 wherein the dextrin contains more than 15% of polymers of adegree of polymerization greater than
 12. 8. The polymer-drug conjugateaccording to claim 7 wherein the dextrin contains more than 50% ofpolymers of DP greater than
 12. 9. The polymer-drug conjugate accordingto claim 1 or 2 wherein a drug loading on the polymer is from 0.5 to99.5 mole %.
 10. The polymer-drug conjugate according claim 1 or 2wherein a targeting group is attached either directly or indirectly tosaid polymer.
 11. The polymer-drug conjugate according to claim 10wherein a ratio of drug to targeting group is from 1:99 to 99:1.
 12. Thepolymer-drug conjugate according to claim 1 or 2 wherein the dextrin iswater soluble or at least forms a suspension in water.
 13. Thepolymer-drug conjugate according to claim 1 or 2 wherein the dextrinused is in the form of unsubstituted dextrin.
 14. The polymer-drugconjugate according to claim 1 or 2 wherein the dextrin is substitutedby at lease one negatively charged, neutral, or positively chargedsubstituent group.
 15. The polymer-drug conjugate according to claim 14wherein the substituent group comprises a sulphate group.
 16. Thepolymer-drug conjugate according to claim 15 wherein the polysaccharidedextrin is substituted with at least one sulphate group per saccharide(glucose) unit.
 17. A pharmaceutical composition comprising thedextrin-drug conjugate of claim 1 or 2 and a pharmaceutically acceptableexcipient or diluent therefor.
 18. The pharmaceutical compositionaccording to claim 17 in the form of an aqueous solution or suspension.19. A method of treating a cancer, comprising administering to a subjecta therapeutically effective amount of the polymer-drug conjugate ofclaim 1 or 2 in the treatment of a cancer in connection with which thedrug is effective.
 20. A method of treating an animal subject, includinga human being, the method comprising treating the animal subject with apharmaceutically effective dose of the dextrin-drug conjugate of claim 1or
 2. 21. The method according to claim 20 wherein the conjugate isadministered intravenously, intraperitoneally, orally, parenterally orby topical application.
 22. A method of preparing a polymer-drugconjugate as claimed in claim 1 or 2, the method comprisingsuccinoylating dextrin and reacting the succinoylated dextrin with thedrug or reactive derivative thereof.
 23. The method according to claim22 wherein the dextrin is dissolved in anhydrous dimethyl formamide. 24.The method according to claim 23 wherein succinoylating dextrincomprises contacting the dissolved dextrin with dimethyl amino pyridineand succinic anhydride.
 25. The method according to claim 24 furthercomprising after succinoylating the dextrin, purging a succinoylateddextrin mixture with an inert gas.
 26. The method according to claim 25wherein succinoylating dextrin is allowed to take place over a prolongedperiod.
 27. The method according to claim 26 wherein said prolongedperiod is at least 12 hours.
 28. The method according to claim 22wherein the succinoylated dextrin is reacted with doxorubicinhydrochloride to form the polymer-drug conjugate.
 29. A conjugate of adextrin and a biologically active agent in which the dextrin iscovalently linked either directly or indirectly to the biologicallyactive agent and the dextrin is a non-cyclic dextrin.
 30. A conjugateaccording to claim 29 wherein the biologically active agent is animaging agent, a diagnostic agent, or a targeting agent.
 31. A conjugateaccording to claim 29 wherein the biologically active agent istyrosinamide.
 32. A conjugate according to claim 29 wherein thebiologically active agent is biotin.